Hexokinases inhibit death receptor-dependent apoptosis on the mitochondria.

Death receptor-mediated apoptosis requires the mitochondrial apoptosis pathway in many mammalian cells. In response to death receptor signaling, the truncated BH3-only protein BID can activate the proapoptotic BCL-2 proteins BAX and BAK and trigger the permeabilization of the mitochondria. BAX and BAK are inhibited by prosurvival BCL-2 proteins through retrotranslocation from the mitochondria into the cytosol, but a specific resistance mechanism to truncated BID-dependent apoptosis is unknown. Here, we report that hexokinase 1 and hexokinase 2 inhibit the apoptosis activator truncated BID as well as the effectors BAX and BAK by retrotranslocation from the mitochondria into the cytosol. BCL-2 protein shuttling and protection from TRAIL- and FasL-induced cell death requires mitochondrial hexokinase localization and interactions with the BH3 motifs of BCL-2 proteins but not glucose phosphorylation. Together, our work establishes hexokinase-dependent retrotranslocation of truncated BID as a selective protective mechanism against death receptor-induced apoptosis on the mitochondria.

Screening for Selective Protein Inhibitors by Using the IANUS Peptide Array.

Finding new road blacks: A peptidic inhibitor of calcineurin (CaN)-mediated nuclear factor of activated T cells (NFAT) dephosphorylation, which is developed through a template-assisted IANUS (Induced orgANisation of strUcture by matrix-assisted togethernesS) peptide array, is cell permeable and able to block the translocation of green fluorescent protein-NFAT fusion protein (GFP-NFAT) into the nucleus after stimulation.

Bax transmembrane domain interacts with prosurvival Bcl-2 proteins in biological membranes.

The Bcl-2 (B-cell lymphoma 2) protein Bax (Bcl-2 associated X, apoptosis regulator) can commit cells to apoptosis via outer mitochondrial membrane permeabilization. Bax activity is controlled in healthy cells by prosurvival Bcl-2 proteins. C-terminal Bax transmembrane domain interactions were implicated recently in Bax pore formation. Here, we show that the isolated transmembrane domains of Bax, Bcl-xL (B-cell lymphoma-extra large), and Bcl-2 can mediate interactions between Bax and prosurvival proteins inside the membrane in the absence of apoptotic stimuli. Bcl-2 protein transmembrane domains specifically homooligomerize and heterooligomerize in bacterial and mitochondrial membranes. Their interactions participate in the regulation of Bcl-2 proteins, thus modulating apoptotic activity. Our results suggest that interactions between the transmembrane domains of Bax and antiapoptotic Bcl-2 proteins represent a previously unappreciated level of apoptosis regulation.

Parvulin 17-catalyzed Tubulin Polymerization Is Regulated by Calmodulin in a Calcium-dependent Manner.

Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function.

N-terminal protein modification by substrate-activated reverse proteolysis.

Although site-specific incorporation of artificial functionalities into proteins is an important tool in both basic and applied research, it can be a major challenge to protein chemists. Enzymatic protein modification is an attractive goal due to the inherent regio- and stereoselectivity of enzymes, yet their specificity remains a problem. As a result of the intrinsic reversibility of enzymatic reactions, proteinases can in principle catalyze ligation reactions. While this makes them attractive tools for site-specific protein bioconjugation, competing hydrolysis reactions limits their general use. Here we describe the design and application of a highly specific trypsin variant for the selective modification of N-terminal residues of diverse proteins with various reagents. The modification proceeds quantitatively under native (aqueous) conditions. We show that the variant has a disordered zymogen-like activation domain, effectively suppressing the hydrolysis reaction, which is converted to an active conformation in the presence of appropriate substrates.

Identification of prolyl oligopeptidase as a cyclosporine-sensitive protease by screening of mouse liver extracts.

Cyclosporine A (CsA) is a cyclic undecapeptide well known for its ability to prevent rejection episodes after organ transplantation via gain-of-function. Therefore, biomedical studies on CsA have been focused on both immunosuppressive properties and binding to the biocatalytically-active immune receptors, the cyclophilins. Much less attention has been spent on effects of cyclosporines on the biological function of other proteins. We used a 9-mer fluorescence-quenched peptide library with defined sequences to identify cyclosporine-sensitive proteolysis in mouse liver extracts. A highly soluble [d-Ser]8-CsA derivative was utilized to avoid drug precipitation at extended incubation times. Analysis of the time courses of proteolysis revealed 15 out of 360 peptide sequences where proteolysis exhibited marked sensitivity to the cyclosporine derivative. As a first example, a collagen-derived substrate was selected from those hits to identify the targeted proteolytic pathway. After purification from mouse liver extracts, prolyl oligopeptidase (EC 3.4.21.26) could be identified as a protease sensitive to submicromolar concentrations of cyclosporines. Surprisingly, in a series of cyclosporine derivatives an inverse relationship was found between the inhibition of prolyl oligopeptidase and inhibition of cyclophilin A.

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials.

BACKGROUND: Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown. METHODS: Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry. RESULTS: MS(2) data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations. CONCLUSIONS: The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials. GENERAL SIGNIFICANCE: The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.

Design of cyclic peptides featuring proline predominantly in the cis conformation under physiological conditions.

Turns are secondary-structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four-residue ß-turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The ßVI-type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c-(-DXaa-Ser-Pro-DXaa-Lys-Pro-) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D-amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis-Pro/cis-Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.

Atomic polarizability dominates the electronic properties of peptide bonds upon thioxo or selenoxo substitution.

The amide bond as peptide linkage plays an important role in protein structure and function. A large number of theoretical and experimental studies have focused on the specific nature of the peptide bond. Little attention, however, has been paid to their chalcogen-substituted congeners, although experimental data on thioamides revealed inconsistencies with the conventional view of amide resonance theory. Here, we employed thioxo and selenoxo substitution to determine experimentally how heavier chalcogens affect the properties of the peptide bond and adjacent atoms. NMR data revealed pronounced deshielding of heteronuclei within a three-bond distance to the chalcogen atom; this indicates an enhanced electron-withdrawing potential of the heavier chalcogens despite their lower electronegativities compared to oxygen. Interestingly, linear correlations were observed between chalcogen atomic polarizability and the chemical shift values of those neighboring heteronuclei as well as several physicochemical properties, such as electronic excitation energy, C-N rotation barrier, dipole moment and amide proton dissociation. We conclude that the chalcogen polarizability, which relates to the charge capacity, is the dominant factor that determines the electronic properties of peptide bonds substituted with heavier chalcogens.

Influence of lithium cations on prolyl peptide bonds.

The influence of lithium cations on the cis/trans isomerization of prolyl peptide bonds was investigated in a quantitative manner in trifluoroethanol (TFE) and acetonitrile, employing NMR techniques. The focus was on various environmental and structural aspects, such as lithium cation and water concentrations, the type of the partner amino acid in the prolyl peptide bond, and the peptide sequence length. Comparison of the thermodynamic parameters of the isomerization in LiCl/TFE and TFE shows a lithium cation concentration dependence of the cis/trans ratio, which saturates at cation concentrations >200 mM. A pronounced increase in the cis isomer content in the presence of lithium cations occurs with the exception of peptides with Gly-Pro and Asp-Pro moieties. The cation effect appears already at the dipeptide level. The salt concentration can considerably be reduced in solvents with a lower number of nucleophilic centers like acetonitrile. The lithium cation effect decreases with small amounts of water and disappears at a water concentration of about 5%. The isomerization kinetics under the influence of lithium cations suggests a weak cation interaction with the carbonyl oxygen of the peptide bond.

The FKBP38 catalytic domain binds to Bcl-2 via a charge-sensitive loop.

FKBP38 is a regulator of the prosurvival protein Bcl-2, but in the absence of detailed structural insights, the molecular mechanism of the underlying interaction has remained unknown. Here, we report the contact regions between Bcl-2 and the catalytic domain of FKBP38 derived by heteronuclear NMR spectroscopy. The data reveal that a previously identified charge-sensitive loop near the putative active site of FKBP38 is mainly responsible for Bcl-2 binding. The corresponding binding epitope of Bcl-2 could be identified via a peptide library-based membrane assay. Site-directed mutagenesis of the key residues verified the contact sites of this electrostatic protein/protein interaction. The derived structure model of the complex between Bcl-2 and the FKBP38 catalytic domain features both electrostatic and hydrophobic intermolecular contacts and provides a rationale for the regulation of the FKBP38/Bcl-2 interaction by Ca(2+).

The action of neutrophil serine proteases on elastin and its precursor.

This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)'. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG.

Suppression of EGFR autophosphorylation by FKBP12.

FK506 binding proteins (FKBPs) represent a subfamily of peptidyl prolyl cis/trans isomerases that can control receptor-mediated intracellular signaling. The prototypic PPIase FKBP12 functionally interacts with EGFR. FKBP12 was shown to inhibit EGF-induced EGFR autophosphorylation with all internal phosphorylation sites equally affected. The inhibition of EGFR catalytic activity is conducted by targeting the EGFR kinase domain. The change of intracellular FKBP12 levels resulted in a change of EGFR autophosphorylation level. Collectively, our results demonstrate that FKBP12 forms an endogenous inhibitor of EGFR phosphorylation directly involved in the control of cellular EGFR activity.

Oligopeptide cyclophilin inhibitors: a reassessment.

Potent cyclophilin A (CypA) inhibitors such as non-immunosuppressive cyclosporin A (CsA) derivatives have been already used in clinical trials in patients with viral infections. CypA is a peptidyl prolyl cis/trans isomerase (PPIase) that catalyzes slow prolyl bond cis/trans interconversions of the backbone of substrate peptides and proteins. In this study we investigate whether the notoriously low affinity inhibitory interaction of linear proline-containing peptides with the active site of CypA can be increased through a combination of a high cis/trans ratio and a negatively charged C-terminus as has been recently reported for Trp-Gly-Pro. Surprisingly, isothermal titration calorimetry did not reveal formation of an inhibitory CypA/Trp-Gly-Pro complex previously described within a complex stability range similar to CsA, a nanomolar CypA inhibitor. Moreover, despite of cis content of 41% at pH 7.5 Trp-Gly-Pro cannot inhibit CypA-catalyzed standard substrate isomerization up to high micromolar concentrations. However, in the context of the CsA framework a net charge of -7 clustered at the amino acid side chain of position 1 resulted in slightly improved CypA inhibition.

Prolyl isomerases show low sequence specificity toward the residue following the proline.

Prolyl isomerases catalyze the cis/trans isomerization of peptide bonds preceding proline. Previously, we had determined the specificity toward the residue before the proline for cyclophilin-, FKBP-, and parvulin-type prolyl isomerases by using proline-containing oligopeptides and refolding proteins as model substrates. Here, we report the specificities of members of these three prolyl isomerase families for the residue following the proline, again in short peptide and in refolding protein chains. Human cyclophilin 18 and parvulin 10 from Escherichia coli show high activity, but low specificity, with respect to the residue following the proline. Human FKBP12 prefers hydrophobic residues at this position in the peptide assays and shows a very low activity in the protein folding assays. This activity was strongly improved, and the sequence specificity was virtually eliminated after the insertion of a chaperone domain into the prolyl isomerase domain of human FKBP12.

Binding specificity of the ectodomain of the parathyroid hormone receptor.

The parathyroid hormone (PTH)1 receptor is a member of the class B G protein-coupled receptor (GPCR) family and regulates bone and mineral metabolism of vertebrates. A truncated highly active parathyroid hormone fragment PTH (1-34) exerts stimulatory effects on the receptor and is used for treatment of osteoporosis. To study the interacting amino acids of the natural peptide ligand PTH (1-84) with the ectodomain of its receptor we used peptide micro arrays on solid cellulose membranes. The amino acids Arg20 and Trp23 within the identified core binding stretch PTH (20-26) were found to be most important for affinity to the ectodomain of PTH1R. Isothermal titration calorimetry and NMR spectroscopy allowed peptide binding studies in solution and verified peptide positions required for high affinity. With this combination of biochemical and biophysical methods we extend former findings on this essential interaction and can now provide a strategy to screen for optimized therapeutic peptides.

The protein-free IANUS peptide array uncovers interaction sites between Escherichia coli parvulin 10 and alkyl hydroperoxide reductase.

The reliable identification of interacting structural elements without prior isolation of interacting proteins can be achieved by using the novel fluorescence resonance energy transfer-coupled IANUS (Induced orgANization of strUcture by matrix-assisted togethernesS) peptide array. Here we report that parvulin 10 (Par10), an abundant Escherichia coli peptidyl prolyl cis/trans isomerase (PPIase), physically interacts with the alkyl hydroperoxide reductase subunit C (AhpC) in bacterial cell extracts, as determined by affinity chromatography and chemical cross-linking experiments. A Par10-negative E. coli strain showed increased sensitivity toward hydrogen peroxide compared to the wild-type strain. The IANUS experiment revealed three segments of the peroxiredoxin AhpC chain as potential Par10 binding partners. Inhibition of the Par10 PPIase activity by the corresponding AhpC-derived peptides as well as NMR data of (15)N-labeled Par10 in the presence of the AhpC(115-132) peptide or full-length AhpC confirmed that the putative Par10 active site is involved in the Par10-AhpC interaction. Moreover, NMR-based docking calculations as well as NOESY exchange peaks between the proline cis and trans isomers revealed the Asp125-Pro126 moiety of the AhpC segment G115-A132 as a substrate for Par10 enzymatic action. On the basis of these data, we conclude that Par10 catalytic activity is involved in the cellular protection against oxidative stress.

New structural aspects of FKBP38 activation.

The human FK506-binding protein 38 (FKBP38) regulates Bcl-2 in neuronal apoptosis. To control Bcl-2 activity, FKBP38 requires a prior interaction with the Ca(2+)-sensor calmodulin (CaM). The resulting FKBP38/CaM complex is unique within the FKBP family. Here, we present novel insights into the structural arrangement of this complex. Chemical shift perturbation analyses of the individual protein domains revealed two separate interaction sites between FKBP38 and CaM. On the one hand, residues Glu303, Tyr307 and Leu311, belonging to the predicted CaM-binding site at the C-terminal end of FKBP38, become embedded in the hydrophobic target protein-binding cleft of the C-terminal CaM lobe. On the other hand, in a second binding interaction, the N-terminal end of the catalytic FKBP38 domain shows surface contacts to the AB and CD loops of CaM as well as the adjacent helices. Furthermore, a Glu-rich region at the non-structured FKBP38 N-terminus features additional contacts to CaM helix A. In combination with previous results, we thus conclude that the FKBP38/CaM complex is constituted by (i) a Ca(2+)-dependent interaction of the CaM-binding motif at the C-terminal end of FKBP38 with the C-terminal CaM lobe and (ii) a Ca(2+)-independent interaction between the N-terminal CaM lobe and the N-terminal region of the catalytic FKBP38 domain.

Rational design of novel peptidic DnaK ligands.

The hsp70 chaperone DnaK from E. coli plays a major role in cellular stress response and is involved in assisted protein folding in vivo. By screening a combinatorial peptide library, we identified several DnaK-specific peptide ligands with nanomolar affinities, which are able to inhibit the secondary amide peptide bond cis/trans isomerase (APIase) activity of DnaK, as well as DnaK/DnaJ/GrpE-assisted refolding of firefly luciferase. Our designed DnaK inhibitors have the capability to penetrate E. coli cells and feature a high protease resistance. Once inside the cell, they physically target DnaK. NMR-based (1)H/(15)N-HSQC experiments furthermore confirmed that the designed peptidic ligands all bind in an identical manner to the conventional peptide-binding site of DnaK. The subsequent blocking of DnaK function apparently results in the observed antibacterial effects on E. coli cells, with minimum inhibitory concentrations in the range of 100 microM.